Various single base or two base point mutations in the human immunodeficiency virus (HIV) genome have been described which are associated with decreased susceptibility of HIV to drug therapy. Larder et al., Science (1989) 246:1155-1158; Kellam et al., Proc. Natl. Acad. Sci. (USA) (1992) 89:1934-1938. The identification of drug resistant strains has been associated with disease progression. One known example of this type of mutation is the two base mutation of amino acid 215 of the HIV-1 reverse transcriptase gene associated with zidovudine (AZT) resistance. Larder et al., supra.
Diagnosis of disease progression, as determined by onset of resistance mutations such as those described above, would be aided by the ability to determine the degree of conversion to the resistant mutant. Current methods, such as "selective PCR" are designed to detect presence or absence of the mutant sequence or wild type/mutant mixture, but are unable to accurately quantify amounts. Boucher et al., Lancet (1990) 336:585-590; Larder et al., AIDS (1991) 5:137-144.
A method for determining the presence or absence of allelic variants in a samples is described in Saiki et al., Nature (1986) 324:163-166; and Farr et al., Proc. Natl. Acad. Sci. (USA) (1987) 85:1629-1633. This method uses the polymerase chain reaction (PCR) technique to amplify a specific region of DNA. The amplified DNA is then used as a target for various radioactively-labelled oligonucleotide probes to identify point mutations and allelic sequence variation. This method has also been used to analyze the allelic genotype of a single sperm at the DNA level. Li et al., Nature (1988) 335:414-417.
An improved method is disclosed herein which provides a method for actually comparing the relative amounts of two variants (e.g., a wild type and a mutant) of a target nucleotide sequence of a target genome in a sample.